1. Field of the Invention
The present invention is directed to peptides that, in association with Class I MHC molecules, form epitopes recognized by cytotoxic T-cells specific for human melanoma, to immunogens comprising said epitopic peptides, and to related compositions, methods and apparatus.
2. Description of the Background Art
Melanoma affects 30,000 new patients per year in the United States. It is a cancer manifested by the unabated proliferation of melanocytes. Eighty percent of melanoma patients are diagnosed during their productive years between the ages of 25 and 65. The incidence of melanoma is rapidly increasing, in 1935 the lifetime risk of developing melanoma was 1:1,500 individuals, at present, the risk has risen to 1:105. It is believed that by the year 2000 the risk of developing melanoma will increase to about 1:70 to 1:90. Early diagnosis and treatment of this disease is crucial. Once a primary tumor becomes metastatic the disease is almost always fatal.
Cytotoxic lymphocyte (CTL) response has been shown to be an important host defense against malignant cells, Rock et al. J. Immunol., (1993), 150:1244.
Lymphocytes isolated from patients having melanoma, when stimulated in vitro with recombinant interleukin-2 (rIL-2) and autologous melanoma cells, develop a melanoma specific cytotoxic response, Vose et al., Nature, (1982), 296:359; Knuth et al., Proc. Natl. Acad. USA, (1984), 81:3511; Slingluff et al., Arch. Surg., (1987), 122:1407; Darrow et al., Cancer, (1988), 62:84; Slingluff et al., J. Natl. Cancer Inst., (1988), 80:1016; Slingluff et al., Ann. Surg., (1989), 210:194; Muul et al., J. Immunol., (1987), 138:989; Van den Eynde et al., Int. J. Cancer, (1989), 44:634; Anichini et al., Int. J. Cancer, (1985), 35:683. The majority of melanoma-specific effector lymphocytes are CD8+ cytotoxic T lymphocytes (CTL) that are restricted by class I Major Histocompatibility Complex (MHC) molecules, Vose et al; Slingluff et al (1988), supra, Hersey et al., Cancer Immunol. Immunother., (1986), 22:15. These characteristics are present whether CTL have been generated from peripheral blood lymphocytes (PBL), lymph node cells, or tumor infiltrating lymphocytes.
The evidence that the CTL response to human melanoma is restricted by class I MHC molecules includes demonstration of cross-reactivity for allogenic melanoma cells that share a restricting class I MHC molecule with the autologous tumor. The HLA-A2 molecule and its variants, of which HLA-A2.1 is by far the most common, is an effective restricting element for the melanoma-specific CTL response. Additionally, melanoma-specific HLA-restricted CTL lyse the majority of A2+ melanomas tested, Darrow et al., J. Immunol., (1989), 142:3329; Wolfel et al., J. Exp. Med., (1989), 170:797; Hom et al., J. Immunother., (1991), 3:153. By demonstrating lysis of A2-melanomas transfected with the A2.1 gene, it has been shown that these transfected melanomas can present the epitopes recognized by A2-restricted melanoma-specific CTL, Kawakami et al., J. Immunol., (1992), 148:638. These results suggest that these CTL recognize A2-restricted epitopes that are shared by the majority of melanomas, although very little is known about the number and identity of their epitopes.
Class I molecules of the Major Histocompatibility Complex (MHC) bind to peptides derived from intracellular pathogens or from proteins expressed in tumor cells, and present them on the cell surface to the host immune system. The mechanism of peptide presentation involves protein synthesis and proteolysis in the cytosol, followed by transport of peptides into the endoplasmic reticulum (ER), through the action of the TAP transporter molecules. Peptides then become associated with newly synthesized class 1 molecules, and the resulting complexes move to the cell surface. Proteins that are membrane associated or secreted contain signal sequences that cause them to be contranslationally transferred into the ER from membrane-bound ribosomes. Such proteins would thus be protected from the action of cytoplasmic proteases. However, since peptide epitopes do arise from such proteins, although their TAP dependent expression is unclear, it has been assumed that the proteolysis to generate these peptide epitopes occurs after these proteins have been aberrantly translated on cytoplasmic ribosomes.
Adoptive transfer of tumor stimulated CTL has been associated with some tumor regressions, Rosenberg et al., N. Eng. J. Med., (1988), 319:1676.
An alternate approach to augmenting the T-cell response to melanoma is the use of a vaccine to stimulate CTL in vivo (active specific immunotherapy). Epitopes for CD8+ CTL are believed to be short, usually 9-residue peptides that bind to a cleft on the surface of the class I MHC molecule, Udaka et al., Cell, (1992), 69:989; VanBleek et al., Nature, (1990), 348:213; Falk et al., J. Exp. Med., (1991), 174:425. These peptides, generated from proteolysis of endogenous proteins in the cytosol, are transported to the endoplasmic reticulum, where they become associated with newly synthesized class I MHC molecules. They are then transported to the cell surface, Elliott et al., Nature, (1990), :348:195. CTL epitopes have been reconstituted in vitro by allowing exogenous peptides to bind to MHC molecules on the cell surface of target cells, Townsend et al., Annu. Rev. Immunol., (1989), 7:601. However, because of the complexity of the peptide mixture associated with class I MHC molecules, Hunt et al., Science, (1992), 255:1261, the definition of individual peptides that comprise specific CTL epitopes has proven extremely difficult.
One method has been to generate genomic or cDNA libraries from tumor cells followed by transfection of progressively smaller subsets of these molecular clones into cells that express the appropriate MHC molecule, but not the tumor specific epitope. Molecular clones that encode T cell epitopes are identified by their ability to reconstitute tumor-specific T cell recognition of the transfected cells. The exact T cell epitope is then identified by a combination of molecular subcloning and the use of synthetic peptides based on the predicted amino acid sequence. See, e.g., P. van der Bruggen et al., Science 254, 1643 (1991); C. Traversari, et al., J. Exp. Med. 176, 1453 (1992); B. Gaugler, et al., ibid. 179, 921 (1994); T. Boon, et al., Annu. Rev. Immunol. 12, 337 (1994); A. B. H. Baker, et al., J. Exp. Med. 179, 1005 (1994); Y. Kawakami, et al., Proc. Natl. Acad. Sci. USA 91, 6458 (1994); P. G. Coulie, et al., J. Exp. Med. 180, 35 (1994); Y. Kawakami, et al., ibid. 180, 347 (1994); V. Brichard, et al., ibid. 178, 489 (1993); T. Wolfei, et al., Eur. J. Immunol. 150, 2955 (1993). Unfortunately, it is possible to inadvertently identify clones that encode cross-reacting peptides that are recognized because of their high level of expression in the transfectants.
By this genetic method HLA-A1 restricted T cell epitope (EADPTGHSY) (SEQ ID NO:99 of a melanoma-associated antigen, MAGE-1, was identified. Traversari, et al., J. Exp. Med., 176:1453–57 (1992). MAGE-1 is expressed in about 20–40% of cancers of several different tissue types, including melanomas, breast cancers, non-small cell lung cancers, head and neck squamous cell cancers, and bladder cancer. It is also found in the normal male testis. The MAGE gene family also includes another member, MAGE-3 for which a homologous HLA-A1 restricted CTL epitope Glu Gal Asp Pro Ile Gly His Leu Tyr (SEQ ID NO:96) only after the first priority date. HLA-A1-restricted CTL epitopes are of limited utility because only a minority of melanomas are HLA-A1+. The function of the MAGE gene products is not known.
The genetic approach has also been used to identify HLA-A2.1-restricted CTL epitopes on tyrosinase. This enzyme is not tumor-specific; it is expressed by normal melanocytes as well as melanoma cells. Tyrosinase is involved in melanin biosynthesis. Autologus CTL recognized tyrosinase-derived HLA-A2-restricted epitopes (Tyr met Asn Gly Thr Met Ser Gln Val (SEQ ID NO:94) Wolfel, et al., Eur. J. Immunol., 24:759–64 (1994). However, these peptides were not recognized by the other CTL lines tested.
Another tissue-specific protein, gp100, is the target of the antibody HMB45, which is specific for melanoma and melanocytes. Based on the correlation between HMB45 activity and recognition by a single TIL-derived HLA-A2-restricted melanoma-specific CTL line, Bakker, et al., J. Exp. Med., 179:1005–9 (1994) established that transfection of cells with the gene for gp100 reconstituted the epitope recognized by this T cell. A subsequent study, using the same T-cell line to screen transfected cDNA libraries also identified the peptide Leu Leu Asp Gly Thr Ala Thr Leu Arg Leu (SEQ ID NO:62) as being sufficient to reconstitute activity. This study was not published prior to Applicants' first priority date. Gp100 is believed to play a role in melanin biosynthesis.
An HLA-A2.1 restricted epitope Ala Ala Gly Ile Leu Thr Val (SEQ ID NO: 97) has also been identified genetically in another melanocytic protein, MART-1 (Melan-A). Kawakami, et al., J. Exp. Med., 180:347–52 (1994) and Proc. Nat. Acad. Sci. USA, 91:3515–19 (1994), and see also Coulie, et al., J. Exp. Med., 180:35–42 (1994).
An alternate approach toward characterization of CTL epitopes is to identify them directly. Naturally occurring peptides associated with MHC molecules on the tumor cells are directly extracted, fractionated by HPLC and used to reconstitute recognition by tumor specific CTL of a non-tumor cell expressing appropriate MHC molecules. Sequencing can be performed by Edman degradation. Mandelboim, et al., Nature, 369:67–71 (1994) (CTL epitope on murine lung carcinoma). However, Applicants pioneered the use of tandem mass spectrometry to evaluate HHC-associated peptides. C. L. Slingluff, et al., J. Immunol. 150, 2955 (1993); D. F. Hunt, et al., Science 255, 1261 (1992); R. A. Henderson, et al., Proc. Natl. Acad. Sci. USA 90, 10275 (1993).
However, when peptides associated with MHC molecules on tumor cells are extracted, a complex mixture, of up to 10,000–20,000 different peptides of similar size (mostly nonamers), is obtained. Within this mixture, only a small number of molecules are likely to correspond to the peptides of interest. Consequently, their isolation and sequencing was extremely difficult. Boon, et al., Ann. Rev. Immunol., 12:337–65 (1994) states, “to our knowledge, the peptide elution method has not yet ensured the identification of a peptide recognized by anti-tumor CTL”. More colorfully, Finn, et al., Curr. Op. Immunol., 5:701–8 (1993) likened the process to “throwing a fish hook into the ocean, hoping to catch the big one”, given, inter alia, the “very low amounts of peptides”.
In the present invention, HLA associated peptides have been extracted, isolated and identified from different melanoma lines. These peptides can be used to reconstitute epitopes for HLA-A2.1- and HLA-A3-restricted melanoma-specific CTL. These peptides and the stimulated CTL may be useful for the in vivo immunotherapeutic treatment of melanoma. Aspects of applicants' invention were described in Cox, et al., Science, 264:716–719 (1994), which was published on Apr. 29, 1994.